Animal cells elaborate a variety of heparan sulfate proteoglycans (HSPGs), which consist of different protein cores and one or more heparan sulfate chains. The HSPGs bind numerous growth factors, cytokines, extracellular matrix components, enzymes and enzyme inhibitors, and they act as receptors or coreceptors for cell signaling, cell attachment, and endocytosis. These interactions depend to a large extent on the composition and the arrangement of sulfated sugars and epimers of uronic acids in the heparan sulfate chains, which in turn depend on the action of various biosynthetic enzymes and regulatory factors. Most of the enzymes involved in heparan sulfate biogenesis have been identified, cloned, expressed as recombinant proteins, studied biochemically, and mutated in cells or model organisms. In contrast, a dearth of information exists regarding the regulatory factors that give rise to the variable composition and binding properties of heparan sulfate. The central hypothesis of this proposal is that genes exist other than those that encode the biosynthetic enzymes, whose expression either modulate the transcription/translation of the enzymes or orchestrate their action to achieve the final composition of heparan sulfate observed in different cell types. Our objective is to search for these regulatory factors through whole genome gene-targeting techniques using the CRISPR/Cas9 genome editing system. Towards this goal, we will (i) adapt high throughput screening assays to identify lentiviral encoded sgRNAs that induce resistance to cytotoxic agents whose action depend on HSPGs (FGF-Saporin and diphtheria toxin); (ii) identify, prioritize, and analyze at the systems level genes tht emerge from the screen, and (iii) target prioritized genes in three different cell lines to understand their mode of action in regulating HSPG expression. Detailed structural studies of HS and binding studies with test ligands also are performed. The significance of this work lies in the potential for discovery of novel regulatory factors involved in HSPG expression. The outcome of these experiments has the wider goal of moving the field into new areas of study. Insight into the mechanisms that cells use to regulate heparan sulfate composition also might lead to novel drug targets for treating human disease associated with alterations in heparan sulfate formation, such as cancer, inflammation and atherosclerosis.